Method of reducing injury to mammalian cells

ABSTRACT

A method of inhibiting the binding between N-methyl-D-aspartate receptors and neuronal proteins in a neuron the method comprising administering to the neuron an effective inhibiting amount of a peptide replacement agent for the NMDA receptor or neuronal protein interaction domain that effect said inhibition of the NMDA receptor neuronal protein. The method is of value in reducing the damaging effect of injury to mammalian cells. Postsynaptic density-95 protein (PSD-95) couples neuronal N-methyl-D-aspartate receptors (NMDARs) to pathways mediating excitotoxicity and ischemic brain damage. This coupling was disrupted by transducing neurons with peptides that bind to modular domains on either side of the PSD-95/NMDAR interaction complex. This treatment attenuated downstream NMDAR signaling without blocking NMDAR activity, protected cultured cortical neurons from excitotoxic insults and dramatically reduced cerebral infarction volume in rats subjected to transient focal cerebral ischemia. The treatment was effective when applied either before, or one hour after, the onset of excitotoxicity in vitro and cerebral ischemia in vivo. This approach prevents negative consequences associated with blocking NMDAR activity and constitutes practical therapy for stroke.

RELATED APPLICATION

[0001] This application is a continuation-in-part application of Ser.No. 09/584,555, filed May 31, 2000.

FIELD OF THE INVENTION

[0002] This invention relates to methods of reducing the damaging effectof an injury to mammalian cells by treatment with compounds which reducethe binding between N-methyl-D-aspartate receptors and neuronalproteins; pharmaceutical compositions comprising said compounds andmethods for the preparation of said pharmaceutical compositions.

BACKGROUND TO THE INVENTION

[0003] Ischemic or traumatic injuries to the brain or spinal cord oftenproduce irreversible damage to central nervous system (CNS) neurons andto their processes. These injuries are major problems to society as theyoccur frequently, the damage is often severe, and at present there arestill no effective pharmacological treatments for acute CNS injuries.Clinically, ischemic cerebral stroke or spinal cord injuries manifestthemselves as acute deteriorations in neurological capacity ranging fromsmall focal defects, to catastrophic global dysfunction, to death. It iscurrently felt that the final magnitude of the deficit is dictated bythe nature and extent of the primary physical insult, and by atime-dependent sequence of evolving secondary phenomena which causefurther neuronal death. Thus, there exists a theoretical time-window, ofuncertain duration, in which a timely intervention might interrupt theevents causing delayed neurotoxicity. However, little is known about thecellular mechanisms triggering and maintaining the processes of ischemicor traumatic neuronal death, making it difficult to devise practicalpreventative strategies. Consequently, there are currently no clinicallyuseful pharmacological treatments for cerebral stroke or spinal cordinjury.

[0004] In vivo, a local reduction in CNS tissue perfusion mediatesneuronal death in both hypoxic and traumatic CNS injuries. Localhypoperfusion is usually caused by a physical disruption of the localvasculature, vessel thrombosis, vasospasm, or luminal occlusion by anembolic mass. Regardless of its etiology, the resulting ischemia isbelieved to damage susceptible neurons by impacting adversely on avariety of cellular homeostatic mechanisms. Although the nature of theexact disturbances is poorly understood, a feature common to manyexperimental models of neuronal injury is a rise in free intracellularcalcium concentration ([Ca²⁺]i). Neurons possess multiple mechanisms toconfine [Ca²⁺]_(i) to the low levels, about 100 nM necessary for thephysiological function. It is widely believed that a prolonged rise in[Ca²⁺]_(i) deregulates tightly-controlled Ca²⁺-dependent processes,causing them to yield excessive reaction products, to activate normallyquiescent enzymatic pathways, or to inactivate regulatory cytoprotectivemechanisms. This, in-turn, results in the creation of experimentallyobservable measures of cell destruction, such as lipolysis, proteolysis,cytoskeletal breakdown, pH alterations and free radical formation.

[0005] The classical approach to preventing Ca²⁺ neurotoxicity has beenthrough pharmacological blockade of Ca²⁺ entry through Ca²⁺ channelsand/or of excitatory amino acid (EAA)—gated channels. Variations on thisstrategy often lessen EAA-induced or anoxic cell death in vitro, lendingcredence to the Ca²⁺-neurotoxicity hypothesis. However, a variety ofCa²⁺ channel- and EAA-antagonists fail to protect against neuronalinjury in vivo, particularly in experimental Spinal Cord Injury (SCI),head injury and global cerebral ischemia. It is unknown whether this isdue to insufficient drug concentrations, inappropriate Ca²⁺ influxblockade, or to a contribution from non-Ca²⁺ dependent neurotoxicprocesses. It is likely that Ca²⁺ neurotoxicity is triggered throughdifferent pathways in different CNS neuron types. Hence, successfulCa²⁺-blockade would require a polypharmaceutical approach.

[0006] As a result of investigations, I have discovered methods ofreducing the damaging effect of an injury to mammalian cells bytreatment with compounds to reduce the binding betweenN-methyl-D-aspartate (NMDA) receptors and neuronal proteins.

PUBLICATIONS

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SUMMARY OF THE INVENTION

[0046] I have found that postsynaptic density-95 protein (PSD-95)couples neuronal N-methyl-D-aspartate receptors (NMDARs) to pathwaysmediating excitotoxicity and ischemic brain damage. This coupling wasdisrupted by transducing neurons with peptides that bind to modulardomains on either side of the PSD-95/NMDAR interaction complex. Thistreatment attenuated downstream NMDAR signaling without blocking NMDARactivity, protected cultured cortical neurons from excitotoxic insultsand dramatically reduced cerebral infarction volume in rats subjected totransient focal cerebral ischemia. The treatment was effective whenapplied either before, or one hour after, the onset of excitotoxicity invitro and cerebral ischemia in vivo. This approach may prevent negativeconsequences associated with blocking NMDAR activity and constitute apractical therapy for stroke.

[0047] It is an object of the present invention to provide in itsbroadest aspect a method of reducing the damaging effect of an injury tomammalian cells.

[0048] In a preferred object, the invention provides pharmaceuticalcompositions for use in treating mammals to reduce the damaging effectof an injury to mammalian tissue.

[0049] The present invention is based on the discovery of aneuroprotective effect against excitotoxic and ischemic injury byinhibiting the binding between N-methyl-D-aspartate (NMDA) receptors andneuronal proteins in a neuron.

[0050] Accordingly, in one aspect the invention provides a method ofinhibiting the binding between N-methyl-D-aspartate receptors andneuronal proteins in a neuron said method comprising administering tosaid neuron an effective inhibiting amount of a peptide replacementagent for the NMDA receptor interaction domain to effect said inhibitionof the interaction with the neuronal protein.

[0051] In a further aspect, the invention provides a method ofinhibiting the binding between N-methyl-D-aspartate receptors andneuronal proteins in a neuron said method comprising administering tosaid neuron an effective inhibiting amount of a peptide replacementagent for the neuronal protein interaction domain to effect saidinhibition of the interaction with the NMDA receptor.

[0052] In a further aspect, the invention provides a method of reducingthe damaging effect of ischemia or traumatic injury to the brain orspinal cord in a mammal, said method comprising treating said mammalwith a non-toxic, damage-reducing, effective amount of a peptidereplacement agent for the NMDA receptor or neuronal protein interactiondomains that inhibit the NMDA receptor neuronal protein interaction.

[0053] Damage to neurons in this specification is meant anoxia,ischemia, excitotoxicity, lack of neurotrophic support, disconnection,and mechanical injury.

[0054] The NMDA agent is, preferably, bindable with proteins containingPDZ domains, and most preferably, is selected from postsynapticdensity-95 proteins, PSD-95, PSD-93 and SAP102.

[0055] I have found that the replacement agent is a tSXV-containingpeptide, preferably KLSSIESDV (SEQ. ID NO: 1).

[0056] The neuronal protein agent is, preferably, bindable withexcitatory amino acid receptors, and most preferably, is selected fromNMDA receptor subunits NR1 and NR2.

[0057] I have found that the replacement agent is a PDZ2-domaincontaining polypeptide, preferably corresponding to residues 65-248 ofPSD-95, encoding the first and second PDZ domains (PDZ1-2) of PSD-95.

[0058] In a yet further aspect the invention provides a pharmaceuticalcomposition comprising a peptide replacement agent for the NMDA receptoror neuronal protein interaction domains that inhibit the NMDA receptorneuronal protein interaction in a mixture with a pharmaceuticallyacceptable carrier when used for reducing the damaging effect of anischemic or traumatic injury to the brain or spinal chord of a mammal;preferably further comprising the cell-membrane transduction domain ofthe human immunodeficiency virus type 1 (HIV-1) Tat protein (YGRKKRRQRRR(SEQ ID No: 2); Tat), or the antennapedia internalisation peptide.

[0059] In a most preferred aspect, the invention provides apharmaceutical composition comprising the peptide KLSSIESDV (SEQ IDNO: 1) or residues 65-248 of PSD-95, encoding the first and second PDZdomains (PDZ1-2) of PSD-95.

[0060] In a further aspect, the invention provides a method ofinhibiting the binding between NMDA receptors and neuronal proteins in aneuron, said method comprising administering to said neuron an effectiveinhibiting amount of an antisense DNA to prevent expression of saidneuronal proteins to effect inhibition of said binding. Preferably, thisaspect provides a method wherein said antisense DNA reduces theexpression of a protein containing PDZ domains bindable to said NMDAreceptor. More preferably, the protein containing PDZ domains isselected from PSD-95, PSD-93 and SAP102.

[0061] In the mammalian nervous system, the efficiency by whichN-methyl-D-aspartate receptor (NMDAR) activity triggers intracellularsignaling pathways governs neuronal plasticity, development, senescenceand disease. I have studied excitotoxic NMDAR signaling by suppressingthe expression of the NMDAR scaffolding protein PSD-95. In culturedcortical neurons, this selectively attenuated NMDAR excitotoxicity, butnot excitotoxicity by other glutamate or Ca²⁺ channels. NMDAR functionwas unaffected, as receptor expression, while NMDA-currents and ⁴⁵Caloading via NMDARs were unchanged. Suppressing PSD-95 selectivelyblocked Ca²⁺-activated nitric oxide production by NMDARs, but not byother pathways, without affecting neuronal nitric oxide synthase (nNOS)expression or function. Thus, PSD-95 is required for the efficientcoupling of NMDAR activity to nitric oxide toxicity and impartsspecificity to excitotoxic Ca²⁺ signaling.

[0062] It is known that calcium influx through NMDARs plays key roles inmediating synaptic transmission, neuronal development, and plasticity(1). In excess, Ca influx triggers excitotoxicity (2), a process thatdamages neurons in neurological disorders that include stroke, epilepsy,and chronic neurodegenerative conditions (3). Rapid Ca²⁺-dependentneurotoxicity is triggered most efficiently when Ca²⁺ influx occursthrough NMDARs, and cannot be reproduced by loading neurons withequivalent quantities of Ca²⁺ through non-NMDARs or voltage-sensitiveCa²⁺ channels (VSCCs) (4). This observation suggests that Ca²⁺ influxthrough NMDAR channels is functionally coupled to neurotoxic signalingpathways.

[0063] Without being bound by theory, I believe that lethal Ca²⁺signaling by NMDARs is determined by the molecules with which theyphysically interact. The NR2 NMDAR subunits, through their intracellularC-terminal domains, bind to PSD-95/SAP90 (5), chapsyn-110/PSD-93, andother members of the membrane-associated guanylate kinase (MAGUK) family(6). NMDAR-bound MAGUKs are generally distinct from those associatedwith non-NMDARs (7). I have found that the preferential activation ofneurotoxic Ca²⁺ signals by NMDARs is determined by the distinctivenessof NMDAR-bound MAGUKs, or of the intracellular proteins that they bind.PSD-95 is a submembrane scaffolding molecule that binds and clustersNMDARs preferentially and, through additional protein-proteininteractions, may link them to intracellular signaling molecules (8).Perturbing PSD-95 would impact on neurotoxic Ca²⁺ signaling throughNMDARs.

[0064] Thus, protein-protein interactions govern the signals involved incell growth, differentiation, and intercellular communication throughdynamic associations between modular protein domains and their cognatebinding partners (20). At excitatory synapses of central neurons,ionotropic glutamate receptors are organized into multi-proteinsignaling complexes within the post-synaptic density (PSD) (21). Aprominent organizing protein within the PSD is PSD-95, a member of themembrane-associated guanylate kinase (MAGUK) family. PSD-95 containsmultiple domains that couple transmembrane proteins such as theN-methyl-D-aspartate subtype of glutamate receptors (NMDAR) to a varietyof intracellular signaling enzymes (21,22). Through its second PDZdomain (PDZ2), PSD-95 binds both the NMDAR 2B subunit (NR2B) andneuronal nitric oxide synthase (nNOS) (22). This interaction couplesNMDAR activity to the production of nitric oxide (NO), a signalingmolecule that mediates NMDAR-dependent excitotoxicity (23). Research hasshown that NMDAR function is unaffected by genetically disrupting PSD-95in vivo (24) or by suppressing its expression in vitro (25).Nonetheless, PSD-95 deletion dissociates NMDAR activity from NOproduction and suppresses NMDAR-dependent excitotoxicity.

[0065] Although NMDARs play an important neurotoxic role inhypoxic/ischemic brain injury (26), blocking NMDAR function may bedeleterious in animals and humans (27-29). Targeting PSD-95 proteintherefore represents an alternative therapeutic approach for diseasesthat involve excitotoxicity that may circumvent the negativeconsequences of blocking NMDAR function. However, mutation orsuppression of PSD-95 is impractical as a therapy for brain injury andcannot be applied after an injury has occurred. Therefore, rather thanalter PSD-95 expression, I questioned whether interfering with theNMDAR/PSD-95 interaction could suppress excitotoxicity in vitro andischemic brain damage in vivo.

BRIEF DESCRIPTION OF THE DRAWINGS

[0066] In order that the invention may be better understood preferredembodiments will now be described by way of example only with referenceto the accompanying drawings wherein:

[0067]FIG. 1a is an immunublot;

[0068]FIG. 1b is a bar chart providing densitometric analysis of PSD-95expression;

[0069]FIG. 1c represents representative phase contrast and propidiumfluorescence images;

[0070]FIG. 1d is a bar chart of NMDA concentration against fraction ofdead cells;

[0071]FIG. 1e is a bar chart of NMDA concentration against Calciumaccumulation.

[0072]FIGS. 2a 1-b 2 represent bar charts of selective activations ofAMPA/Kainate receptors with Kainate (2 a 1 and 2-a 2); and loadings withVscc's (2-b 1) and calcium loading (2-b 2).

[0073]FIGS. 3a-d represent immunoblots (3 a); NMDA dose-response curves(3 b); NMDA current density measurements (3 c); and current/time graph(3 d) dialyzed with hucifer yellow;

[0074]FIG. 4 bar charts (4 a; 4 c-4 f) and immunoblot 4 b of effect onnNOS expression in cultures are hereinafter better described andexplained;

[0075]FIG. 5. (A) Shows the hypothesis: The NMDAR/PSD-95 complex may bedissociated by peptides encoding either to the C-terminus of NR2 or thesecond PDZ domains of PSD-95 (B) Fluorescence of cultures treated withTat-38-48-dansyl and Tat-NR2B9c dansyl. (C) Time course fluorescenceafter Tat-NR2B9c-dansyl application (D) Effect of peptides onco-immunoprecipitation of PSD-95 with NR2B

[0076]FIG. 6. Effect of Tat-NR2B9c on (A-C) electrophysiologicalfunction of neurons (D) NMDA-evoked ⁴⁵Ca²⁺ uptake in cortical cultures.(E) NMDA-evoked cGMP production in cortical cultures. (F) NMDA-evokedexcitotoxicity in cortical cultures.

[0077]FIG. 7. (A) Detection of Tat-NR2B9c-dansyl in the mouse brain 1 hafter intraperitoneal injection (B) Composite neurological scores (seetext) during and 24 h after MCAo. (C) Effect of Pre-treatment withTat-NR2B9c on (i) total infarct area and volume (inset), and (ii)cortical infarct area and volume (inset) after transient MCAo.

[0078]FIG. 8. (A) Neuroprotective effects of post-treatment in culturedcortical neurons post-treated with Tat-NR2B9c or pTat-PDZ1-2 (B)Composite neurological scores (see text) during and 24 h after MCAo. (C)Effect of post-treatment with Tat-NR2B9c on (i) total infarct area andvolume (inset), and (ii) cortical infarct area and volume (inset) aftertransient MCAo.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

[0079] Methods:

[0080] Cultured cortical neurons were prepared by standard techniques(4,9) and switched to serum-free media at 24 h [Neurobasal with B27supplement (Gibco)]. The AS ODN corresponded to nucleotides 435-449 ofmouse PSD-95/SAP90 mRNA (GeneBank Acc. No. D50621). Filter-sterilizedphosphodiester AS SE, and MS ODNs (5 μM) were added in culture mediumduring feedings at 4,6,8 and 10 days after plating. Cultures were usedfor all experiments (FIGS. 1-4) on day 12. ODN sequences exhibited nosimilarity to any other known mammalian genes (BLAST search (10)).

[0081] Immunoblotting was done as described in ref. “26”. Tissue washarvested and pooled from 2 cultures/lane. The blotted proteins wereprobed using a monoclonal anti-PSD-95 mouse IgG1 (Transduction Labs,1:250 dilution), polyclonal anti PSD-93 (1:1000 dilution) and antiSAP-102 (1:2000 dilution) rabbit serum antibodies (Synaptic SystemsGmbH), a monoclonal anti NR1 mouse IgG2a (PharMingen Canada, 1:1000dilution) or a monoclonal anti nNOS (NOS type I) mouse IgG2a(Transduction Labs, 1:2500 dilution). Secondary antibodies were sheepanti-mouse, or donkey anti-rabbit Ig conjugated to horseradishperoxidase (Amersham). Immunoblots for PSD-95 were obtained for allexperiments (FIGS. 1-4) from sister cultures, and all gels quantifiedusing an imaging densitometer (Bio-Rad GS-670).

[0082] cGMP determinations were performed 10 min after challenging thecultures with NMDA, kainate, or high-K (FIGS. 4c-e) with the BiotrakcGMP enzymeimmunoassay system according to the kit manufacturer'sinstructions (Amersham). Staining for NADPH diaphorase (FIG. 4b) wasdone as described in ref. 12.

[0083] Electrophysiology. Whole cell patch-clamp recordings in thecultured neurons were performed and analyzed as described in ref. 13.During each experiment a voltage step of −10 mV was applied from holdingpotential and the cell capacitance was calculated by integrating thecapacitative transient. The extracellular solution contained (in mM):140 NaCl, 5.4 KCl, 1.3 CaCl₂, 25 HEPES, 33 glucose, 0.01 glycine, and0.001 tetrodotoxin (pH=7.3-7.4, 320-335 mOsm). A multi-barrel perfusionsystem was employed to rapidly exchange NMDA containing solutions. Thepipette solution contained (in mM): 140 CsF, 35 CsOH, 10 HEPES, 11 EGTA,2 tetraethylammonium chloride (TEA), 1 CaCl₂, 4 MgATP, pH 7.3 at 300mOsm. Lucifer yellow (LY; 0.5% w/v) was included in the pipette forexperiments in FIG. 3d.

[0084] Excitotoxicity and Ca²⁺ accumulation measurements were performedidentically to the methods described and validated in refs. 4 and 14. Weused measurements of propidium iodide fluorescence as an index of celldeath, and of radiolabelled ⁴⁵Ca²⁺ accumulation for Ca²⁺ loaddeterminations in sister cultures on the same day. Experimentalsolutions were as previously described (4). Ca²⁺ influx waspharmacologically channeled through distinct pathways as follows: ToNMDARs by applying NMDA (×60 min) in the presence of both CNQX (ResearchBiochemicals Inc) and nimodipine (Miles Pharmaceuticals), to non-NMDARsby applying kainic acid (×60 min or 24 h) in the presence of both MK-801(RBI) and nimodipine, and to VSCCs using 50 mM K+ solution (×60 min)containing 10 mM Ca²⁺ and S(−)-Bay K 8644, an L-type channel agonist(300-500 nM; RBI), MK-801 and CNQX. Antagonist concentrations were (inμM): MK-801 10, CNQX 10, nimodipine 2. All three antagonists were addedafter the 60 min agonist applications for the remainder of allexperiments (24 h). A validation of this approach in isolating Ca²⁺influx to the desired pathway in our cortical cultures has beenpublished (4).

[0085] Whole cell patch-clamp recordings in the cultured neurons wereperformed and analyzed as described in Z. Xiong, W. Lu, J. F. MacDonald,Proc Natl Acad Sci USA 94, 7012 (1997). During each experiment a voltagestep of −10 mV was applied from holding potential and the cellcapacitance was calculated by integrating the capacitative transient.The extracellular solution contained (in mM): 140 NaCl, 5.4 KCl, 1.3CaCl₂, 25 HEPES, 33 glucose, 0.01 glycine, and 0.001 tetrodotoxin(pH=7.3-7.4, 320-335 mOsm). A multi-barrel perfusion system was employedto rapidly exchange NMDA containing solutions. The pipette solutioncontained (in mM): 140 CsF, 35 CsOH, 10 HEPES, 11 EGTA, 2tetraethylammonium chloride (TEA), 1 CaCl₂, 4 MgATP, pH 7.3 at 300 mOsm.Lucifer yellow (LY; 0.5% w/v) was included in the pipette forexperiments in FIG. 3D.

[0086] Data analysis: data in all figures were analyzed by ANOVA, with apost-hoc Student's t-test using the Bonferroni correction for multiplecomparisons. All means are presented with their standard errors.

[0087] In greater detail:

[0088]FIG. 1, shows increased resilience of PSD-95 deficient neurons toNMDA toxicity in spite of Ca²⁺ loading. A. Immunoblot showingrepresentative effects of sham (SH) washes, and PSD-95 AS, SE and MSODNs, on PSD-95 expression. PC: positive control tissue from purifiedrat brain cell membranes. Asterisk: non-specific band produced by thesecondary antibody, useful to control for protein loading and blotexposure times. B. Densitometric analysis of PSD-95 expression pooledfrom N experiments. Asterisk: different from other groups, one-wayANOVA, F=102, p<0.0001. ODNs were used at 5 μM except where indicated(AS 2 μM). C. Representative phase contrast and propidium iodidefluorescence images of PSD-95 deficient (AS) and control (SE) cultures24 h after a 60 min challenge with 30 μM NMDA. Scale bar: 100 μm. D.Decreased NMDA toxicity at 24 h in PSD-95 deficient neurons followingselective NMDAR activation×60 min (n=16 cultures/bar pooled from N=4separate experiments). Asterisk: differences from SE, MS and SH(Bonferroni t-test, p<0.005). Death is expressed as the fraction of deadcells produced by 100 μM NMDA in sham-ODN-treated controls (validated in4,14). E. No effect of PSD-95 deficiency on NMDAR-mediated Ca²⁺ loading(n=12/bar, N=3; reported as the fraction of ⁴⁵Ca²⁺ accumulationachievable over 60 min in the sham controls by 100 μM NMDA, whichmaximally loads the cells with calcium (4).

[0089]FIG. 2, shows thatPSD-95 deficiency does not affect toxicity andCa²⁺ loading produced by activating non-NMDARs and Ca²⁺ channels.Cultures were treated with SH washes or AS or SE ODNs as in FIG. 1. A.Selective activation of AMPA/kainate receptors with kainate in MK-801(10 μM) and nimodipine (NIM; 2 μM) produces toxicity over 24 h (A1)irrespective of PSD-95 deficiency, with minimal ⁴⁵Ca²⁺ loading (A2). B.Selective activation of VSCCs produces little toxicity (B1), butsignificant ⁴⁵Ca²⁺ loading (B2) that is also insensitive to PSD-95deficiency. n=4 cultures/bar in all experiments.

[0090]FIG. 3, shows that there is no effect of perturbing PSD-95 onreceptor function. A. Immunoblots of PSD-95 ODN-treated cultures probedfor PSD-95, NR1, PSD-93, and SAP-102 using specific antibodies. PC:positive control tissue from purified rat brain cell membranes. B. NMDAdose-response curves and representative NMDA currents (inset) obtainedwith 3-300 μM NMDA. C. NMDA current density measurements elicited with300 μM NMDA (AS: n=18; SE: n=19; SH: n=17; one-way ANOVA F=1.10,p=0.34), and analysis of NMDA current desensitization.I_(ss)=steady-state current; I_(peak)=peak current. AS: n=15; SE: n=16;SH: n=16 (ANOVA,, F=0.14, p=0.87). Time constants for current decay wereAS: 1310±158 ms; SE, 1530±185 ms; SH: 1190±124 ms (ANOVA, F=1.22,p=0.31). D. Currents elicited with 300 μM NMDA in neurons dialyzed withLY (insert) and 1 mM tSXV or control peptide.

[0091]FIG. 4, shows the effect of coupling of NMDAR activation to nitricoxide signaling by PSD-95. A. L-NAME protects against NMDA toxicity(n=4, N=2). Asterisk: difference from 0 μM L-NAME (Bonferroni t-test,p<0.05). B. No effect of SH and of PSD-95 AS and MS ODNs on nNOSexpression in cultures (immunoblot) and on NADPH diaphorase staining inPSD-95 AS and SE-treated neurons. PC: positive control tissue frompurified rat brain cell membranes. C. Effect of isolated NMDARactivation on cGMP formation (n=12 cultures/bar pooled from N=3 separateexperiments) D,E. Effects of VSCC activation (n=8/bar, N=2), andAMPA/kainate receptor activation (n=4/bar, N=1) on cGMP formation. Datain C-E are expressed as the fraction of cGMP produced in SE-treatedcultures by 100 μM NMDA. Asterisk: differences from both SH and SEcontrols (Bonferroni t-test, p<0.0001). F. Sodium nitroprusside toxicityis similar in PSD-95 AS, SE and SH treated cultures.

[0092] PSD-95 expression was suppressed in cultured cortical neuronsto<10% of control levels, using a 15-mer phosphodiester antisense (AS)oligodeoxynucleotide (ODN) (FIGS. 1A,B) Sham (SH) washes, sense (SE) andmissense (MS) ODNs (9) had no effect. The ODNs had no effect on neuronalsurvivability and morphology as gauged by viability assays, hereinbelow, and phase-contrast microscopy (not shown).

[0093] To examine the impact of PSD-95 on NMDAR-triggeredexcitotoxicity, ODN-treated cultures were exposed to NMDA (10-100 μM)for 60 min, washed, and either used for ⁴⁵Ca²⁺ accumulationmeasurements, or observed for a further 23 h. Ca²⁺ influx was isolatedto NMDARs by adding antagonists of non-NMDARs and Ca²⁺ channels (4).NMDA toxicity was significantly reduced in neurons deficient in PSD-95across a range of insult severities (FIGS. 1C,D; EC₅₀: AS: 43.2±4.3; SE:26.3±3.4, Bonferroni t-test, p<0.005). Concomitantly however, PSD-95deficiency had no effect on Ca²⁺ loading into identically treated sistercultures (FIG. 1E). Therefore, PSD-95 deficiency induces resilience toNMDA toxicity despite maintained Ca²⁺ loading.

[0094] I next examined whether the increased resilience to Ca²⁺ loadingin PSD-95 deficient neurons was specific to NMDARs. Non-NMDAR toxicitywas produced using kainic acid (30-300 μM), a non-desensitizingAMPA/kainate receptor agonist (15), in the presence of NMDAR and Ca²⁺channel antagonists (4). Kainate toxicity was unaffected in PSD-95deficient in neurons challenged for either 60 min (not shown) or 24 h(FIG. 2A1). Non-NMDAR toxicity occurred without significant ⁴⁵Ca²⁺loading (FIG. 2A2), as>92% of neurons in these cultures expressCa²⁺-impermeable AMPA receptors (4). However, Ca²⁺ loading throughVSCCs, which is non-toxic (4) (FIG. 2B1), was also unaffected by PSD-95deficiency (FIG. 2B2). Thus, suppressing PSD-95 expression affectsneither toxicity nor Ca²⁺ fluxes triggered through pathways other thanNMDARs.

[0095] Immunoblot analysis (11) of PSD-95 deficient cultures revealed noalterations in the expression of the essential NMDAR subunit NR1, nor oftwo other NMDAR-associated MAGUKs, PSD-93 and SAP-102 (FIG. 3A). Thisindicated that altered expression of NMDARs and their associatedproteins was unlikely to explain reduced NMDA toxicity in PSD-95deficiency (FIGS. 1C,D). Therefore, I examined the possibility thatPSD-95 modulates NMDAR function. NMDA currents were recorded using thewhole-cell patch technique (16) (FIG. 3B). PSD-95 deficiency had noeffect on passive membrane properties, including input resistance andmembrane capacitance [Capacitance: AS 55.0±2.6 pF (n=18 ); SE 52.7±3.2pF (n=19); SH 48.1±3.4 pF (n=17; ANOVA, F=1.29, p=0.28)]. Whole-cellcurrents elicited with 3-300 μM NMDA were also unaffected. Peak currentswere AS: 2340±255 pA (n=18); SE: 2630±276 (n=19); SH: 2370±223 (n=17)(FIG. 3B, inset; one-way ANOVA, F=0.43, p=0.65). NMDA dose-responserelationships also remained unchanged (FIG. 3B; EC₅₀ AS: 16.1±0.8 μM(n=7); SE: 15.5±2.1 (n=6); SH: 15.9±2.9; one-way ANOVA, F=0.02, p=0.98),as were NMDA current density and desensitization (FIG. 3C).

[0096] To further examine the effect of PSD-95 binding on NMDARfunction, a 9 aa peptide, KLSSIESDV (SEQ ID NO: 1) corresponding to theC-terminal domain of the NR2B subunit characterized by the tSXV motif(6) was injected into the neurons. At 0.5 mM, this peptide competitivelyinhibited the binding of PSD-95 to GST-NR2B fusion proteins (6), and wastherefore predicted to uncouple NMDARs from PSD-95. Intracellulardialysis of 1 mM tSXV or control peptide, CSKDTMEKSESL (SEQ ID NO: 3)(6) was achieved through patch pipettes (3-5 MΩ) also containing thefluorescent tracer Lucifer Yellow (LY). This had no effect on NMDAcurrents over 30 min despite extensive dialysis of LY into the cell somaand dendrites (FIG. 3D). Peak current amplitudes were tSXV: 2660±257 pA(n=9), control: 2540±281 pA (n=10; t₍₁₇₎=0.31, p=0.76).

[0097] The data is consistent with that obtained from recently generatedmutant mice expressing a truncated 40K PSD-95 protein that exhibitedenhanced LTP and impaired learning (17). Hippocampal CA1 neurons inPSD-95 mutants exhibited no changes in NMDAR subunit expression andstoichiometry, cell density, dendritic cytoarchitecture, synapticmorphology, or NMDAR localization using NR1 immunogold labeling ofasymmetric synapses. NMDA currents, including synaptic currents, werealso unchanged (16). I also found no effects of PSD-95 deficiency onNMDAR expression, on other NMDAR associated MAGUKs, nor on NMDA-evokedcurrents. In addition, NMDAR function gauged by measuring NMDA-evoked⁴⁵Ca²⁺-accumulation was unaffected. Thus, the neuroprotectiveconsequences of PSD-95 deficiency must be due to events downstream fromNMDAR activation, rather than to altered NMDAR function.

[0098] The second PDZ domain of PSD-95 binds to the C-terminus of NR2subunits and to other intracellular proteins (8). Among these is nNOS(18), an enzyme that catalyzes the production of nitric oxide (NO), ashort-lived signaling molecule that also mediates Ca²⁺-dependent NMDAtoxicity in cortical neurons (12). Although never demonstratedexperimentally, the NMDAR/PSD-95/nNOS complex was postulated to accountfor the preferential production of NO by NMDARs over other pathways (8).To determine whether NO signaling plays a role in NMDA toxicity in thepresent cultures, we treated the cells with N^(G)-nitro-L-argininemethyl ester (L-NAME), a NOS inhibitor (12). L-NAME protected theneurons against NMDA toxicity (FIG. 4A), indicating the possibility thatsuppressing PSD-95 might perturb this toxic signaling pathway.

[0099] The effect of suppressing PSD-95 expression on NO signaling andtoxicity was examined using cGMP formation as a surrogate measure of NOproduction by Ca²⁺-activated nNOS (20,21). PSD-95 deficiency had noimpact on nNOS expression (FIG. 4B), nor on the morphology (FIG. 4B) orcounts of NADPH diaphorase-staining (12) neurons (SH: 361±60, SE:354±54, AS: 332±42 staining neurons/10 mm coverslip, 3coverslips/group). However, in neurons lacking PSD-95 challenged withNMDA under conditions that isolated Ca²⁺ influx to NMDARs (4), cGMPproduction was markedly attenuated (>60%; FIG. 4C, one-way ANOVA,p<0.0001). Like inhibited toxicity (FIGS. 1,2), inhibited cGMP formationin neurons lacking PSD-95 was only observed in response to NMDA. It wasunaffected in neurons loaded with Ca²⁺ through VSCCs (FIG. 4D), evenunder high neuronal Ca²⁺ loads matching those attained by activatingNMDARs (compare FIGS. 1E and 2B2) (4). nNOS function therefore, wasunaffected by PSD-95 deficiency. AMPA/kainate receptor activation failedto load the cells with Ca²⁺ (FIG. 2A2), and thus failed to increase cGMPlevels (FIG. 4E). Our findings indicate that suppressing PSD-95selectively reduces NO production efficiency by NMDAR-mediated Ca²⁺influx, but preserves NO production by Ca²⁺ influx through otherpathways.

[0100] Bypassing nNOS activation with NO donors restored toxicity inneurons lacking PSD-95. The NO donors sodium nitroprosside (12) (FIG.4F; EC₅₀ 300 μM) and S-nitrosocysteine (17) (not shown) were highlytoxic, irrespective of PSD-95 deficiency. Thus, reduced NMDA toxicity inPSD-95 deficient cells was unlikely to be caused by altered signalingevents downstream from NO formation.

[0101] Suppressing PSD-95 expression uncoupled NO formation from NMDARactivation (FIG. 4C), and protected neurons against NMDAR toxicity(FIGS. 1C,D) without affecting receptor function (FIGS. 1E, 3A-D), bymechanisms downstream from NMDAR activation, and upstream fromNO-mediated toxic events (FIG. 4F). Therefore, PSD-95 imparts NMDARswith signaling and neurotoxic specificity through the coupling ofreceptor activity to critical second messenger pathways. These resultshave broader consequences, as NMDAR activation and NO signaling are alsocritical to neuronal plasticity, learning, memory, and behavior(1,18,19). Thus, these data provide experimental evidence for amechanism by which PSD-95 protein may govern important physiological andpathological aspects of neuronal functioning.

[0102]FIG. 5 shows the utility of Tat-peptides in dissociating theNMDAR/PSD-95 interaction (A) The hypothesis: The NMDAR/PSD-95 complex(left panel) may be dissociated using Tat peptides fused either to theC-terminus of NR2B (Tat-NR2B9c; middle) or to the first and second PDZdomains of PSD-95 (pTat-PDZ1-2; right), thus reducing the efficiency ofexcitotoxic signaling via Ca²⁺-dependent signaling molecules (B)Intracellular accumulation of Tat-NR2B9c-dansyl (10 μM) but not controlpeptide (Tat-38-48-dansyl; 10 μM) was observed 30 min after applicationto cortical neuronal cultures using confocal microscopy (excitation: 360nm, emission: >510 nm; representative of 5 experiments). Fluorescence ofcultures treated with Tat-38-48-dansyl was similar to background (notshown). (C) Time course of Tat-NR2B9c-dansyl (10 μM) fluorescence afterapplication to cortical cultures at room temperature (symbols: mean±S.Eof 4 experiments). Inset: fluorescence images from representativeexperiment (D) Tat-NR2B9c, but not control peptides (see text), inhibitsthe co-immunoprecipitation of PSD-95 with NR2B in rat forebrain lysates(Left: Representative gel; Right: means±S.E of 4 experiments, ANOVA,F=6.086, *p=0.0041).

[0103] In more detail, a conserved tSXV motif at the C-terminus of theNR2B subunit is critical for binding to the PDZ2 domain of PSD-95. Ihypothesized that interfering with this interaction might disrupt thecoupling between NMDARs and PSD-95. This might be achieved by theintracellular introduction of exogenous peptides that bind to either theNR2B or the PDZ2 interaction domains (FIG. 5A). To this end I used apeptide comprised of the nine C-terminal residues of NR2B (KLSSIESDV;NR2B9c (SEQ ID NO: 1)), which is anticipated to bind the PDZ2 domain ofPSD-95. As an alternative means to interfere with the NMDAR/PSD-95interaction I constructed a protein comprised of residues 65-248 ofPSD-95 encompassing the first and second PDZ domains (PDZ1-2), whichcontains the principal binding domain in PSD-95 for the C-terminus ofNR2B. NR2B9c or PDZ1-2 on their own did not enter cells (not shown) andtherefore, I fused each to a peptide corresponding to the cell-membranetransduction domain of the HIV-1-Tat protein (YGRKKRRQRRR (SEQ ID NO:2); Tat) to obtain a 20 amino acid peptide (Tat-NR2B9c) and the fusionprotein pTat-PDZ1-2. pTat-PDZ1-2 and pTat-GK fusion proteins weregenerated by insertion of PSD95 residues 65-248 encoding the PDZ 1 and2, and residues 534-724 encoding the guanylate kinase-like domains,respectively, into pTAT-HA plasmids (generous gift of S. Dowdy,Washington University, St.Louis, Mo.). Fusion proteins contain a 6XHis-tag, the protein transduction domain of HIV-1 Tat and ahemagglutinin-tag N-terminal to the insert. Plasmids were transformedinto BL21(DE3)LysS bacteria (Invitrogen) and recombinant proteins wereisolated under denaturing conditions on a Nickle-His column(Amersham-Pharmacia). These are anticipated to transduce cell membranesin a rapid, dose-dependent manner independent of receptors andtransporters (30).

[0104] To determine whether Tat-NR2B9c was able to transduce intoneurons, I conjugated the fluorophore dansyl chloride to Tat-NR2B9c andto a control peptide comprised of HIV-1-Tat residues 38-48 (KALGISYGRKK(SEQ ID NO: 4); Tat38-48) outside the Tat transduction domain (31).

[0105] Electrophysiological Recordings were made in 400 μm Hippocampalslices from 20-36 day old Sprague-Dawley rats perfused at roomtemperature with ACSF containing (in mM) 126 NaCl, 3 KCl, 2 MgCl₂, 2CaCl₂, 1.2 KH₂PO₄, 26 NaHCO₃ and 10 glucose and bubbled with95%O₂/5%CO₂. Whole-cell recordings of CA1 neurons were performed usingthe “blind” method with an Axopatch-1D amplifier (Axon Instruments,Foster City, Calif.) at holding potential −60 mV. Pipettes (4-5 MΩ) werefilled with solution containing (mM): 135 CsCl, 2 MgCl₂, 0.1 CaCl₂, 0.5EGTA, 10 HEPES, 4 Mg-ATP, 0.2 GTP, and 5 QX-314, pH 7.4, 310 mOsm. Fieldpotentials were recorded with glass micropipettes (2-4 MΩ) filled withACSF placed in the stratum radiatum 60-80 μm from the cell body layer.Synaptic responses were evoked by stimulation (0.05 ms) of the Schaffercollateral-commissural pathway with a bipolar tungsten electrode in thepresence of bicuculline methiodide (10 μM). For I_(NMDA) recording, Mg²⁺was removed from and 20 μM CNQX was added in ACSF. Following 10-20 minbase line recordings of EPSCs, I_(NMDA) and fEPSPs, Tat-peptides wereapplied in ACSF and recordings were continued for 30 min thereafter.

[0106] I bath applied these to cultured cortical neurons and observedtheir fluorescence by confocal microscopy. Neurons treated withTat-NR2B9c-dansyl (10 μM) exhibited fluorescence in their cytoplasm andprocesses, indicating intracellular peptide delivery (FIG. 5B, left).Sister cultures treated with Tat38-48-dansyl (10 μM) exhibited onlybackground fluorescence, indicating no observable peptide uptake in theabsence of the Tat transduction domain (FIG. 5B, right).Tat-NR2B9c-dansyl was detectable in the neurons within 10 min of thestart of the application and the peptide accumulated to a maximum levelover the next 20 min (FIG. 5C). This level was maintained until thedansyl-Tat-NR2B9c was washed from the bath and the peptide remaineddetectable within the neurons for more than 5 hours thereafter.Therefore, the Tat transduction domain was able to act as a carrier forNR2B9c and the Tat-NR2B9c fusion peptide remained in neurons for manyhours after being applied extracellularly.

[0107] To determine whether Tat-NR2B9c may disrupt the interactionbetween NMDARs and PSD-95 I made use of rat brain proteins preparedunder weakly denaturing conditions known to permit the NMDAR/PSD-95interaction. Adult (7-8W) wistar rat forebrains were removed andhomogenized in ice-cold buffer (0.32M Sucrose, 0.1 mM Na3VO4, 0.1 mMPMSF, 0.02M PNPP, 0.02M glycerol phosphate, and 5 ug/ml each ofantipain, aprotinin, and leupeptin). Homogenates were centrifuged at 800gr for 10 min at 4° C. The supernatants were combined and centrifuged at11,000 g at 4 degree for 20 min and the pellet (P2) was resuspended inhomogenization buffer. P2 membranes were adjusted 200 ug protein/90 ulwith homogenization buffer with a final concentration of 1% DOC and 0.1%Triton X-100. The proteins were incubated with Tat-NR2B9c or with one ofthree controls: Tat38-48, the Tat transduction sequence conjugated totwo alanine residues (Tat-AA), or a Tat-NR2B9c peptide in which theC-terminal tSXV motif contained a double point mutation(Tat-KLSSIEADA;Tat-NR2BAA) rendering it incapable of binding PSD-95. I immunoprecipatedNMDARs, together with associated proteins, with an antibody thatselectively recognizes NR2B. The proteins were separated by SDS-PAGE andprobed with anti-PSD-95 or anti-NR2B antibodies¹⁶ NR2B was precipitatedfrom rat forebrain extracts using a polyclonal rabbit anti-NR2B antibodygenerated against the C-terminal region encompassing amino acid residues935-1,455 of the NR2B protein. Proteins were then separated on 8%SDS-PAGE gels and probed with monoclonal anti-NR2B (Clone 13,Transduction Laboratories) or anti PSD-95 antibodies (Clone 7E3-1B8,Affinity Bioreagents. Inc). Detection of proteins was achieved usingHRP-conjugated secondary antibodies and enhanced chemiluminescence. Ifound that Tat-NR2B9c reduced the co-immunoprecipitation of PSD-95 withNR2B. On average the optical density signal was reduced by 37.6±8.2% ascompared with controls (FIG. 5D). In contrast, none of the three controlpeptides reduced the co-immmunoprecipitation of PSD-95 with NR2B. Thus,I conclude that Tat-NR2B9c disrupts the interaction between NMDARs andPSD-95 and that this is dependent upon an intact PDZ binding motif inthe peptide.

[0108]FIG. 6 shows neuroprotection and reduction of NO signaling byTat-peptides without affecting NMDAR function (A) Effect of Tat-NR2B9c(50 nM) on field excitatory post-synaptic currents (fEPSC) in CA1neurons in acute hippocampal slices. (B) Effect of 50 nM Tat-NR2B9c orTat-38-48 (control) on whole-cell excitatory post synaptic currents(EPSC). (C) Effect of Tat-NR2B9c on the NMDA component of the EPSCisolated pharmacologically by applying the AMPAR antagonist CNQX, andconcomitant removal of extracellular Mg²⁺. (D) Effect of 50 nMTat-NR2B9c treatment on NMDA-evoked ⁴⁵Ca²⁺ uptake in cortical cultures.Tat-peptides were bath-applied 1 h prior to the NMDA application. (E)Effect of 50 nM Tat-NR2B9c treatment on NMDA-evoked cGMP production incortical cultures. Asterisk: differences from control and Tat-NR2B-AA ateach NMDA concentration (Bonferroni t-test, p<0.01). (F) Decreasedexcitotoxicity at 20 h at all NMDA concentrations in cultured corticalneurons pre-treated with 50 nM Tat-NR2B9c or pTat-PDZ1-2 for 1 h.Asterisk: differences from control, Tat-NR2B-AA and pTat-GK at each NMDAconcentration (Bonferroni t-test, p<0.005). Right panels: Representativephase contrast and propodium iodide fluorescence images of cultures 20 hafter challenge with 100 μM NMDA with and without Tat-NR2B9c treatment.Bars in (D), (E) and (F) indicate the mean±S.E. for 12 cultures in 3separate experiments.

[0109] In more detail, as NMDAR-mediated synaptic responses are notaltered by the loss of PSD-95 (24) I predicted that Tat-NR2B9c would notaffect the function of NMDARs. This was tested by examining the effectof Tat-NR2B9c on NMDAR-mediated currents and on NMDA-evoked uptake of⁴⁵Ca²⁺. Bath-applying Tat-NR2B9c (50 nM) to acute rat hippocampal sliceshad no effect on synaptic responses of CA1 neurons evoked by stimulationof the Schaffer collateral-commissural pathway (FIG. 6A) nor on patchrecordings of the total excitatory post-synaptic currents (EPSC)recorded in CA1 neurons, (FIG. 6B) nor on the pharmacologically isolatedAMPA (not shown) or NMDA components of the EPSC (FIG. 6C). Moreover,using cortical cultures I found that pre-treating cultures withTat-NR2B9c or with pTat-PDZ1-2 (each at 50 nM) did not alter the uptakeof ⁴⁵Ca²⁺ produced by applying NMDA (FIG. 6D); CNQX (10 μM) andnimodipine (2 μM) were present in the extracellular solution in theseand all subsequent experiments using cultured neurons so as to isolatesignaling and thereby preventing secondary activation of AMPARs or ofvoltage-gated Ca²⁺ channels, respectively (25,32,33).

[0110] As the function of NMDARs was unaffected by administeringTat-NR2B9c, I next determined whether this peptide altered signalingevents downstream of NMDAR activation. To this end I examinedstimulation of nNOS, as a key downstream signaling enzyme that mediatesthe neurotoxic effects of NMDAR activation⁵. I measured NMDA-evokedchanges in the levels of guanosine 3′,5′-monophosphate (cGMP) as asurrogate measure of NO production by NMDAR stimulated nNOSactivity^(7,20). Cultured cortical neurons were pre-treated for 1 h withTat-NR2B9c (50 nM), the non-interacting Tat-NR2B-AA (50 nM) or with shamwashes and challenged with NMDA (0-1000 μM) in the presence of CNQX andnimodipine as above. NMDA produced a concentration-dependent increase incGMP that was significantly suppressed (average of 39.5±6.7%) bypre-treating the cultures with Tat-NR2B9c (FIG. 6E). In contrast,NMDAR-stimulated elevation of cGMP was unaffected by pre-treatment withTat-NR2B-AA. Thus, Tat-NR2B9c, but not a mutant peptide incapable ofinteracting with PSD-95, depressed NMDAR-evoked stimulation of NO-cGMPsignaling.

[0111] Although Tat-NR2B9c and pTat-PDZ1-2 did not affect NMDARfunction, Tat-NR2B9c was shown to interfere with NMDAR/PSD-95 bindingand to suppress downstream NO signaling. Thus, I predicted thatTat-peptide treatment should enhance neurons' resilience to NMDAtoxicity. To test this I pre-treated cortical neuronal cultures withTat-peptides (50 nM) for 1 h, then applied NMDA (0-100 μM) for 1 hfollowed by a 20 h observation period (FIG. 6F, inset). Control neuronswere treated with sham washes, or with the non-interacting controlTat-NR2BAA. In cultures treated with Tat-NR2B9c, cell death wassignificantly reduced at all concentrations tested (FIG. 6F) whereaspre-treatment with Tat-NR2B-AA had no effect on cell death. Thus,NMDAR-stimulated neurotoxicity is suppressed by pre-treatment withTat-NR2B9c, suppression that is lost by mutating the PSD-95 bindingregion of the peptide.

[0112] If Tat-NR2B9c suppresses NMDA excitotoxicity by interfering withthe binding of NR2B to PSD-95 then interfering with this binding by analternative means should also suppress the toxicity. I testedpTat-PDZ1-2, predicted to interfere with PSD-95 binding to NR2B andwhich permeates into the cells (not shown), though without effect onNMDA-evoked Ca²⁺ accumulation (FIG. 6D). Pre-treating the cultures withpTat-PDZ1-2 attenuated the neurotoxicity of NMDA to a similar degree asTat-NR2B9c (FIG. 6F). As a control, I made and used pTat-GK, a Tatfusion protein containing residues 534-724 of PSD-95 comprising thecarboxyl-terminal guanylate-kinase homology domain that lacks enzymaticactivity²¹. pTat-GK, which is devoid of the necessary domains to bindNR2B, had no effect on the NMDA-evoked cell death (FIG. 6F). Thus,interfering with the NMDAR/PSD-95 interaction using peptides that targeteither side of the interaction reduces in vitro excitotoxicity producedby NMDAR activation.

[0113]FIG. 7 shows neuroprotection by Tat-NR2B9c pretreatment in-vivo.(A) Detection of Tat-NR2B9c-dansyl but not Tat38-48-dansyl in the cortexof C57BL/6 mouse brain 1 h after intraperitoneal injection (0.5 μmoletotal dose). Fluorescence of brains from animals treated withTat-38-48-dansyl was similar to background (not shown). (B) Compositeneurological scores (see text) during and 24 h after MCAo. (C)Pre-treatment with 3 nmole/g Tat-NR2B9c but not mutated Tat-NR2B-AA orsaline (control) significantly reduced (i) total infarct area and volume(inset), ANOVA; F=7.3, p<0.005 and (ii) cortical infarct area and volume(inset), ANOVA; F=8.35, p<0.005 measured 24 h after transient MCAo. (n=6animals per group; symbols and bars indicate mean±S.E). Infarct volumewas calculated by analyzing the infarct area in 8 stereotacticcoordinates of the brain as shown at right inset.

[0114] Agents that block NMDAR activity were initially deemed aspromising neuroprotectants for stroke and other neurological disordersinvolving excitotoxic mechanisms, but were later shown to be deleteriousor ineffective in animal and human studies (27,28,29). However,Tat-peptides that target the NMDAR/PSD-95 interaction protect againstNMDA toxicity without blocking NMDARs. Therefore I reasoned thattreatment with Tat-NR2B9c in vivo could serve as an improvement on NMDAblockers in the treatment of ischemic brain damage.

[0115] Before testing this I determined whether Tat-NR2B9c could bedelivered into the brain in the intact animal. I injected 25 g C57BL/6mice intraperitoneally with a 500 μmole dose of eitherTat-NR2B9c-dansyl, or with Tat38-48-dansyl as a non-transducing control.40 μm cryostat coronal brain sections taken 1 h after injection²² wereexamined for peptide uptake using dansyl fluorescence detection byconfocal microscopy. The mice were perfused with fixative solution (3%paraformaldehyde, 0.25% glutaraldehyde, 10% sucrose, 10 U/mL heparin inSaline) 1 hour after peptide injection. Brains were removed, frozen in2-methylbutane at −42° C. and 40□ m sections were cut using a LeitzKryostat. Brain sections from animals injected with Tat-NR2B9c exhibitedstrong fluorescence in the cortex (FIG. 7A, right), and in all otherareas examined (hippocampus, striatum; not shown), whereas signal fromcontrols remained at background levels (FIG. 7A, left). Similar resultswere obtained using intravenous injection in rats (not shown). Thus,Tat-NR2B9c enters the brain upon peripheral administration.

[0116] Next, I examined whether pretreatment with Tat-peptides wouldreduce stroke damage. Experiments were carried out in adult maleSprague-Dawley rats subjected to transient middle cerebral arteryocclusion (MCAO) for 90 minutes by the intraluminal suture method(36,37). Animals were fasted overnight and injected with atropinesulfate (0.5 mg/kg IP). After 10 minutes anesthesia was induced with3.5% halothane in a mixture of nitrous oxide and oxygen (Vol. 2:1) andmaintained with 0.8% halothane. Rats were orally intubated, mechanicallyventilated, and paralyzed with pancuronium bromide (0.6 mg/kg IV). Bodytemperature was maintained at 36.5-37.5° C. with a heating lamp.Polyethylene catheters in the femoral artery and vein were used tocontinuously record blood pressure and to sample blood for gas and pHmeasurements. Transient MCAO was achieved for 90 min by introducing apoly-L-lysine-coated 3-0 monofilament nylon suture (Harvard Apparatus)into the circle of Willis via the internal carotid artery, effectivelyoccluding the middle cerebral artery. This produces an extensiveinfarction encompassing the cerebral cortex and basal ganglia. Animalswere pretreated with either saline, the Tat-NR2B-AA control, or withTat-NR2B9c by a single intravenous bolus injection 45 min prior to MCAO(3 nMoles/g). Physiological parameters (body temperature, bloodpressure, blood gases) were monitored and maintained throughout theexperiment (Table 1). All experimental manipulations and analyses ofdata were performed by individuals blinded to the treatment groups. Theextent of cerebral infarction was measured 24 h after MCAO onset (FIG.7C inset). The postural reflex test (38), and the forelimb placing test(39) were used to grade neurological function on a scale of 0 to 12(normal=0; worst=12) during MCAO (at 50 minutes) and 24 h thereafter.

[0117] Pretreatment with Tat-NR2B9c produced a trend toward improvementin 24 h neurological scores in animals treated with Tat-NR2B9c (FIG.7B). Moreover, the treatment reduced the volume of total cerebralinfarction by 54.6±11.27% as compared with stroke volume in controls(FIG. 7C_(i); ANOVA, F=7.289, p=0.0048). This effect was largelyaccounted-for by a 70.7±11.23% reduction in cortical infarction (FIG.7C_(ii), ANOVA, F=8.354, p=0.0027), which is thought to be largelycaused by NMDAR-dependent mechanisms.

[0118] A treatment for stroke with a single-bolus drug injection wouldbe most therapeutically valuable if effective when given after the onsetof ischemia. I thus first evaluated whether treatment with Tat-peptidescould be neuroprotective when applied post-insult in vitro.

[0119]FIG. 8 shows neuroprotection by post-treatment with Tat-NR2B9cin-vitro and in-vivo (A) Decreased excitotoxicity at 20 h in culturedcortical neurons post-treated with 50 nM Tat-NR2B9c or pTat-PDZ1-2 at 1h after NMDA application. Bars indicate the mean±S.E. for 12 cultures in3 separate experiments. Asterisk: differences from control, Tat-NR2B-AAand pTat-GK at each NMDA concentration (Bonferroni t-test, p<0.005).Right panels: Representative phase contrast and propodium iodidefluorescence images of cultures 24 h after challenge with 100 μM NMDAwith and without Tat-NR2B9c treatment. (B) Composite neurological scores(see text) during and 24 h after MCAo. Asterisk: difference from controland Tat-NR2B-AA (ANOVA; F=17.25, p<0.0001). (C) Post-treatment with 3nmole/g Tat-NR2B9c (9 animals) but not mutated Tat-NR2B-AA (8 animals)or saline controls (10 rats) significantly reduced (i) total infarctarea and volume (inset), ANOVA; F=12.0, p<0.0005 and (ii) corticalinfarct area and volume (inset), ANOVA; F=12.64, p=0.0001 as measured 24h after transient MCAo. Symbols and bars indicate mean±S.E (D).Representative appearance of H&E stained rat brain sections from whichthe infarct areas were analyzed.

[0120] Cultured cortical neurons were exposed to an NMDA challenge(0-100 μM) for 1 h and were then treated with the Tat-peptides (all at50 nM) described in the pre-treatment study (FIG. 6F). Cell death wasgauged 20 h thereafter (FIG. 8A—inset). Post-treatment with Tat-NR2B9cor with pTat-PDZ1-2 significantly reduced the vulnerability of neuronsto NMDA toxicity as compared with control cultures post-treated withsham washes, with Tat-NR2BAA, or with pTat-GK (FIG. 8A). Thus, whenadministered 1 hr after the start of the NMDA insult each of the Tatfusion constructs that target the NMDAR/PSD-95 interaction significantlyreduced neuronal cell death in vitro.

[0121] Finally, I examined whether treatment with Tat-NR2B9c couldattenuate ischemic neuronal damage in-vivo when given after strokeonset. A post-treatment study was conducted in which the rats weresubjected to transient MCAO for 90 minutes as before, but theintravenous saline or Tat-peptide bolus (Tat-NR2B9c or Tat-NR2B-AA; 3nMole/g) was injected 1 h after MCAO onset (FIG. 8C—inset). Infarctionvolume and neurological outcome measurements were performed at timesidentical to the pre-treatment study. Body temperature, blood pressureand blood gases were monitored throughout the 24 h experiment andmaintained equivalent between groups (Table 2).

[0122] Post-treatment with Tat-NR2B9c, but not with Tat-NR2B-AA orsaline, resulted in animals exhibiting a significant improvement in 24 hneurological scores as compared with controls (FIG. 8B; ANOVA, F=17.25,p<0.0001). Most strikingly, post-treatment with Tat-NR2B9c reduced thevolume of total cerebral infarction by 67.0±3.75% as compared withstroke volume in controls (FIG. 8C_(i); ANOVA, F=11.99, p=0.0002).Similar to the previous study, this reduction was accounted-for by a86.97±4.38% reduction in cortical infarction volume (FIG. 8C_(ii), 4D;ANOVA, F=12.64, p<0.0001).

[0123] The aforesaid description demonstrates that introducing intocells an exogenous peptide containing the C-terminal nine amino acids ofthe NR2B NMDAR subunit has profound effects on signaling pathwaysdownstream of NMDAR activation, on in vitro excitotoxicity, and on invivo ischemic brain damage. The effects of this peptide are lost bymutating amino acids that are essential for mediating PDZ binding toPSD-95. In addition, a protein comprising PDZ1-2 of PSD-95 shares theeffects of the NR2B C-terminal peptide. Together these findings implythat the downstream signaling from NMDARs that leads to negativeconsequences for neuronal viability may be interrupted by interferingwith the interaction between NR2B and PSD-95.

[0124] I have discovered that the strategy of treating neurons withTat-fusion peptides is effective in reducing vulnerability toexcitotoxicity in vitro and stroke damage in vivo. As this occurswithout affecting NMDAR activity then adverse consequences of blockingNMDARs are not expected. Efficacy after the insult onset suggests thattargeting the NMDAR/PSD-95 interaction is a practical future strategyfor treating stroke. It is also likely that targeting otherintracellular proteins using the same approach could be used to modulateadditional signaling mechanisms mediated by protein-protein interactionsthat lead to other human diseases. TABLE 1 Physiological Variables inPre-Treatment MCAO Study Control TAT-NR2BAA TAT-NR2B9c PhysiologicalVariables (n = 6) (n = 6) (n = 6) Before anesthesia Body weight, g 269 ±6  273 ± 7  271 ± 5  Before MCAo (45 min) Body Temperature, ° C. 36.7 ±0.07 36.7 ± 0.17 36.6 ± 0.21 MABP, mmHg 119 ± 4  115 ± 5  120 ± 9 Before MCAo (30 min) Body Temperature, ° C. 36.8 ± 0.08 36.5 ± 0.12 36.7± 0.19 MABP, mmHg 107 ± 3  110 ± 4  76 ± 5* Blood gases PH 7.44 ± 0.027.44 ± 0.02 7.44 ± 0.02 PO2, mmHg 104 ± 3  110 ± 7  123 ± 8  PCO2, mmHg39.6 ± 1.3  39.1 ± 1.4  38.1 ± 1.4  Before MCAo (15 min) BodyTemperature, ° C. 36.9 ± 0.11 36.6 ± 0.15 36.7 ± 0.20 MABP, mmHg 111 ±6  115 ± 5  90 ± 6* During MCAo (5 min) Body Temperature, ° C. 36.9 ±0.03 36.6 ± 0.17 36.7 ± 0.16 MABP, mmHg 132 ± 6  135 ± 7  112 ± 9  Bloodgases PH 7.44 ± 0.02 7.44 ± 0.02 7.44 ± 0.02 PO2, mmHg 118 ± 3  109 ± 4 112 ± 6  PCO2, mmHg 39.2 ± 0.6  39.6 ± 0.5  41.0 ± 1.3  During MCAo (15min) Body Temperature, ° C. 36.9 ± 0.09 36.7 ± 0.15 36.8 ± 0.23 MABP,mmHg 116 ± 9  111 ± 6  98 ± 6  After MCAo (15 min) Body Temperature, °C. 36.9 ± 0.09 36.8 ± 0.08 36.8 ± 0.12 After MCAo (24 hr) BodyTemperature, ° C. 36.6 ± 0.14 37.0 ± 0.25 36.5 ± 0.14 Body weight, g 238± 6  244 ± 6  250 ± 5 

[0125] TABLE 2 Physiological Variables in Post- Treatment MCAO StudyControl TAT-NR2BAA TAT-NR2B9c Physiological Variables (n = 10) (n = 8)(n = 9) Before anesthesia Body weight, g 314 ± 4  301 ± 5  306 ± 7 Before MCAo (15 min) Body Temperature, ° C. 36.9 ± 0.07 36.7 ± 0.07 36.6± 0.07 MABP, mmHg 103 ± 4  103 ± 6  103 ± 5  Blood gases PH 7.43 ± 0.017.45 ± 0.01 7.43 ± 0.02 PO2, mmHg 113 ± 4  113 ± 4  105 ± 4  PCO2, mmHg39.4 ± 1.0  37.9 ± 1.1  40.1 ± 1.0  During MCAo (15 min) BodyTemperature, ° C. 36.9 ± 0.07 36.7 ± 0.11 37.0 ± 0.07 MABP, mmHg 120 ±5  121 ± 5  119 ± 8  Blood gases PH 7.44 ± 0.01 7.46 ± 0.01 7.43 ± 0.01PO2, mmHg 113 ± 3  108 ± 2  111 ± 4  PCO2, mmHg 39.3 ± 0.7  48.0 ± 1.2 39.8 ± 0.9  During MCAo (60 min) Body Temperature, ° C. 37.1 ± 0.21 37.0± 0.31 36.7 ± 0.11 MABP, mmHg 146 ± 5  149 ± 4  143 ± 5  During MCAo (65min) Body Temperature, ° C. 37.1 ± 0.16 37.0 ± 0.29 36.9 ± 0.08 MABP,mmHg 134 ± 6  136 ± 5  137 ± 4  After MCAo (15 min) Body Temperature, °C. 37.0 ± 0.09 36.9 ± 0.23 36.8 ± 0.08 MABP, mmHg 128 ± 6  116 ± 4  119± 4  After MCAo (24 hr) Body Temperature, ° C. 36.6 ± 0.14 36.7 ± 0.2736.4 ± 0.24 Body weight, g 276 ± 3  276 ± 6  279 ± 8 

[0126] Although this disclosure has described and illustrated certainpreferred embodiments of the invention, it is to be understood that theinvention is not restricted to those particular embodiments. Rather, theinvention includes all embodiments which are functional or mechanicalequivalence of the specific embodiments and features that have beendescribed and illustrated.

I claim:
 1. A method of inhibiting the binding betweenN-methyl-D-aspartate receptors and neuronal proteins in a neuron in vivosaid method comprising administering to said neuron an effectiveinhibiting amount of a peptide replacement agent for a NMDA receptor orneuronal protein interaction domain to effect inhibition of the NMDAreceptor neuronal protein.
 2. A method as defined in claim 1 whereinsaid neuron is damaged.
 3. A method of reducing the damaging effect ofischemia or traumatic injury to the brain or spinal chord in a mammal,said method comprising treating said mammal with a non-toxic,damage-reducing, effective amount of a peptide replacement agent for theNMDA receptor or neuronal protein interaction domain to effect saidinhibition of the NMDA receptor neuronal protein.
 4. A method as definedin claim 3 wherein said mammal is under the influence of neuronal celldamage.
 5. A method as defined in claim 1 wherein said NMDA receptor isbindable with proteins containing PDZ domains.
 6. A method as defined inclaim 5 wherein said protein containing PDZ domains is PSD-95.
 7. Amethod as defined in claim 5 wherein said protein containing PDZ domainsis PSD-93.
 8. A method as defined in claim 5 wherein said proteincontaining PDZ domains is SAP102.
 9. A method as defined in claim 5wherein said protein containing PDZ domains is SAP97.
 10. A method asdefined in claim 1 wherein said protein containing PDZ domains is atSXV-containing peptide.
 11. A method as defined in claim 10 whereinsaid agent is KLSSIESDV (SEQ ID NO: 1).
 12. A method as defined in claim1 wherein said neuronal protein is bindable with excitatory amino acidreceptors
 13. A method as defined in claim 1 wherein said neuronalprotein is bindable with NMDA receptors
 14. A method as defined in claim1 wherein said neuronal protein is bindable with AMPA receptors
 15. Amethod as defined in claim 1 wherein said neuronal protein is bindablewith metabotropic glutamate receptors
 16. A method as defined in claim 1wherein said agent is a peptide encoding a PDZ-binding domain of aprotein containing PDZ domains.
 17. A method as defined in claim 16wherein said agent is residues 65-248 of PSD-95.
 18. A method ofcontrolling the concentration of Ca²⁺-dependent signaling molecules inthe vicinity of ion channel pores of cells in vivo to prevent thediffusion of toxic amounts of said Ca²⁺ influx to prevent the triggeringof neurotoxic phenomena, said method comprising administering aneffective, non-toxic amount of a peptide replacement agent for the NMDAreceptor or neuronal protein interaction domain that effect saidinhibition of the NMDA receptor neuronal protein.
 19. A method asdefined in claim 18 wherein said agent is a tSXV-containing peptide. 20.A method as defined in claim 19 wherein said agent is KLSSIESDV (SEQ IDNO: 1).
 21. A method as defined in claim 18 wherein said agent is apeptide encoding a PDZ-binding domain of a protein containing PDZdomains.
 22. A method as defined in claim 21 wherein said agent isresidues 65-248 of PSD-95.